Recently the drug development has been more and more aimed at search for drug forms enabling targeted action of active substance primarily at the site of the required therapeutic effect. The targeted active forms of drug find application with the substances whose side effects may lead to damage of healthy parts of the organism. The most topical concern is the danger of damaging healthy tissues and organs, endangering patients treated with cytostatics. The utilization of polymer materials, in particular water-soluble polymers as carriers for targeted transport of drugs is one of the significant possibilities of solution to the mentioned problem. Attachment of a cytostatic to water-soluble polymer with a covalent bond makes it possible to significantly enhance the solubility of drugs poorly soluble in water and to reduce pronouncedly their toxicity. The molecular weights of polymer-drug conjugates prevents rapid elimination of the drug from the organism by glomerular filtration, thus prolonging significantly its time of circulation in blood and also its total dwell time in the organism thus leading to enhanced biological utilizability of the drug. In addition, high molecular weight of the polymer conjugate leads to its higher accumulation in solid tumours due to the effect of enhanced permeability and retention (EPR) [Maeda 2000].
If a cancerostatic is attached to polymer carrier, this effect can be utilized for its targeted accumulation in tumour. In the last two decades, a number of systems have been developed that are based on utilization of the EPR effect for targeting drugs such as polymer micelles, liposomes or water-soluble polymer conjugates. Polymer micelles, in contrast to soluble polymer systems, are usually prepared by assembling amphiphilic diblock copolymers into HMW structures forming colloid solutions. The drug is attached to micelles mostly in their hydrophobic cores by physical (hydrophobic) interactions or covalent bonds [Kataoka 2001, Bae 2003, Bronich 1999]. In contrast to micelles of soluble systems accumulating in solid tumours, polymers are dispersed molecularly in aqueous media, adopting usually a shape of random coil, in which the drug is in contact with hydrophilic polymer. Many types of conjugates of cancerostatics with soluble polymers were studied, in which the drug was attached by hydrolytically labile ionic bonds or covalent bonds prone to enzymatic or common chemical hydrolysis. The mentioned systems can release the cancerostatic in its active form in tumor tissue or also in a specific way directly in the tumour cell. Among water-soluble systems, polymer conjugates prepared on the basis of N-(2-hydroxypropyl)methacrylamide (HPMA) copolymers belong to the most important. A number of them are actively directed into tumours by an attached targeting structure (antibodies, hormones and oligopeptides). [Duncan 1985, {hacek over (R)}íhová 2000, Kope{hacek over (c)}ek 2001, Mrkvan 2005].
One of the main drawbacks preventing the use of HPMA copolymers as drug carriers in human medicine is their non-cleavable carbon chain and the associated limits of molecular weights utilizable for preparation of polymer carriers of molecular weights to those lower than 40,000-50,000 (below the renal threshold of the organism). Polymers of high molecular weights are not eliminated from the organism effectively and sufficiently and hence their use as drugs would lead to their accumulation in the organism. If a pronounced EPR effect, i.e. significant accumulation in solid tumours, it is necessary to work with polymers, including HPMA copolymers, with molecular weights highly above the exclusion limits [Seymour 1995, Noguchi 1998]. Therefore, it is important that the molecular weight of polymer carrier is sufficiently high, but the polymer can be degraded, after release of the active component, to fragments, which can be eliminated from the organism, e.g., by glomerular filtration. Recently, we have patented structures and biological activities of drugs using HPMA polymers and their associates with molecular weights above the limit of renal filtration. These were graft polymer carriers [Etrych patent CZ 298 945 B6, Etrych 2008] and polymer micelles [Chytil patent CZ PV 2006-207] based on HPMA copolymers, in which a cancerostatic was attached to polymers with covalent bonds, which are enzymatically or just hydrolytically labile, and, possibly, polymer chains were linked to graft structures through biodegradable, enzymatically and/or reductively cleavable spacers, namely oligopeptide GlyPheLeuGly or a disulfide bridge. An advantage of the systems containing cancerostatic doxorubicin (Dox) as active component was an enhanced antitumour activity verified in mouse models compared with original polymer systems containing the same drug. A disadvantage of the mentioned systems is a relatively broad distribution of molecular weights (polydispersity index In ranging from 3 to 4), hence a not well defined system with a limited possibility of obtaining high molecular weights exceeding 200,000 g/mol. The subject of the invention is the structure and synthesis of a new HMW polymer drug with a narrow distribution of molecular weights (In˜1.5−2.5) and with a defined biodegradable skeleton characterized by enhanced accumulation of the cytostatic in tumour and, also, after intracellular degradation, by elimination of the polymer carrier from the organism. Their structure emanates from a dendritic central part, bearing on dendritic branches polymer grafts containing a covalently attached drug. By choice of the number of grafts and their size, it is possible to obtain high molecular weights of the polymer (up to 1,400,000 g/mol). Similar systems were described previously [Wang 2000], a principial difference between the described system and that of the subject of the invention and the previously published systems consists in that the previously described systems do not have a simply degradable polymer skeleton and the drug is attached only through the enzymatically degradable GlyPheLeuGly oligopeptide sequence. For modification of dendrimer branch ends, poly(ethylene glycol) (PEG) [Gajbhiye 2009, Bai 2009] was often used. The main task of PEG in these systems was hindering potentially toxic amino groups of dendrimers; PEG does not serve here as a carrier of biologically active molecules. Similarly to the preceding case, the systems are not biodegradable. If they should not accumulate in the organism, their utilization is limited to low molecular weights up to 50,000 g/mol.
Use of dendrimers with other, often biodegradable polymers has been mentioned in the patent literature. Poly(amino acid)s were used for preparation of dendrimer-poly(amino acid) conjugates [Li patent WO03055935], where the conjugate was prepared by polymerization of N-carboxyanhydrides of a-amino acids with dendrimer initiators. In this case, the poly(amino acid) grafts bear a significant negative charge (poly(glutamic acid) and poly(aspartic acid)), a positive charge (polyarginine, polyhistidine, polylysine) or are hydrophobic and hence insoluble in body fluids. A drug, e.g. paclitaxel, is bound to the polymer through a covalent ester bond. Due to a high charge density on the system surface or its considerable hydrophobicity and insolubility, undesirable interactions with various tissues and accumulation in the organism (e.g. kidneys) may occur as it is typical, e.g., of negatively charged poly(amino acid)s [Rypá{hacek over (c)}ek 1982]. Nor attachment of a drug to the polymer with a covalent ester bond without a biodegradable spacer guarantees the release of the drug in its original active form. In systems utilizing poly(amino acid) grafts, only partial degradation of poly(glutamic acid) is proved, in which cleavage of a broad spectrum of low-molecular-weight fragments and amino acid derivatives of the drug, which are not necessarily biologically active, happens. Degradation of such systems is documented in literature only in a model system containing cathepsin B and the data on their degradation in vivo are incomplete.
An advantage of the system according to the present invention are the polymer grafts formed by an inert, uncharged, water-soluble polymer, non-interacting with the organism, based on HPMA copolymer, to which the drug is attached through intracellularly degradable spacers enabling controlled release of active drug in target cells and tissues. Another advantage of the system according to the invention is the unequivocally proved intracellular reductive or enzymatic degradation of the carrier skeleton to polymer products of inert water-soluble HPMA copolymer which can be eliminated from the organism by glomerular filtration. Thus the elimination of HMW polymer carrier from the organism, after transporting a cancerostatic to the tumour site is guaranteed.
In literature, other systems are described, in which hydrophilic chains of poly[N-(2-hydroxypropyl) methacrylamide], poly(N-vinylpyrrolidone), poly(ethylene glycol methacrylate, poly(N-isopropylacrylamide), polyacrylic acid, poly(methacrylic acid, poly(2-aminoethyl methacrylate], poly[N-(3-aminopropyl)methacrylamide], and poly[2-(dimethylamino)ethyl methacrylate] were attached to linear or star poly(E-caprolactone), poly(L-lactide), poly(D-lactide), poly(DL-lactide), and poly(glycolic acid) [Lele patent U.S. Pat. No. 7,018,655]. The authors show that the thus prepared systems form HMW systems are utilizable in drug transport. The systems form micelles or nanoparticles in solution thanks to the amphiphilic nature of their structure, containing, e.g., hydrophobic poly(ε-caprolactone) and a HPMA-based polymer. The drug in these systems is then sorbed by hydrophobic interaction, on the hydrophobic core of the system, which does not enable controlled release of the drug in dependence on external conditions and cannot prevent the drug release in the course of transport. Moreover, this system is in most cases loaded with problems similar to those of the system described in a patent [Li patent WO03055935], i.e. a surface charge and undesirable interactions in the organism.
In contrast to the above-mentioned system, that is, according to the invention, soluble in water and body fluids, without a possibility of aggregation, bearing a drug attached through a biodegradable spacer, which enables controlled release of the drug in target cells or tumour tissue.
In literature, also the systems are described utilizing poly(amidoamine) (PAMAM) dendrimers as carriers, e.g. for anti-inflammation drugs [Kurtoglu 2009], in which the drug (N-acetylcysteine) is bound to a dendrimer through a reductively cleavable disulfide bridge. In this system, PAMAM dendrimer serves as a carrier. Hence, due to the PAMAM, it is difficult to obtain a high molecular weight, which is important for passive targeting into the tumour tissue.